Solutions to Practice Problems for Recombinant DNA, Session 5: Agarose Gel

Electrophoresis, DNA Sequencing, and PCR

Question 1

You make a cDNA library by cloning the cDNA fragments into a unique EcoRI restriction site in the vector.
You identify a recombinant vector that you believe has the gene of interest.

a) How can you use the EcoRI restriction enzyme to tell you if the gene has been inserted?
You would inoculate media with some cells from the colony that you have identified. You would allow
many cells to grow, and then isolate the vector from these cells. Once you have the vector DNA, you
would digest it with EcoRI, and separate the resulting DNA fragments on an agarose gel. If your vector
had a cDNA inserted into the EcoRI site, you will see at least two bands on your gel.

Suppose you find that the gene of interest is in the vector, but now you want a restriction map of the
recombinant plasmid. You take three individual samples of the plasmid and digest one sample with
EcoRI, the second sample with HindIII, and the third sample with both EcoRI and HindIII. Then you run
the digested DNA on an agarose gel to see the fragments.

5.3
4.1
3.0
2.7

2.3

1.5
1.1

0.4

b) Considering that the cDNA fragment is smaller than the vector

Circle the fragments on the gel that contain all or part of the cDNA.

Draw the restriction map of this recombinant plasmid.

Eco RI

1.1 kb Hind III

0.4 kb Eco RI

3.0 kb

2.3 kb

Hind III
Question 2

a) Given the sequence below, design primers, each 16 nucleotides long, which would allow you to
generate many copies of a PCR product that was 400 base pairs long

GGACCGCGGGGCAGGATTGCTCCGGGCTGTTTCATGACTTGTCAGGTGGGATGACTTGGATGGAAAAGTAGAAGGTCATG
1 +---------+---------+---------+---------+---------+---------+---------+---------
CCTGGCGCCCCGTCCTAACGAGGCCCGACAAAGTACTGAACAGTCCACCCTACTGAACCTACCTTTTCATCTTCCAGTAC

GGGTGGCCAACTTGGGCGAGAAAAGGTATATAAAGGTCTCTTGCTCCCATCAACTGCCTCAAAAGTAGGTATTCCAGCAG
81 +---------+---------+---------+---------+---------+---------+---------+---------
CCCACCGGTTGAACCCGCTCTTTTCCATATATTTCCAGAGAACGAGGGTAGTTGACGGAGTTTTCATCCATAAGGTCGTC

ATCAGACAACGTCAGGTGGGAGGACTTGGACGGAAAAGTAGAAGGTCAAGACCAACCTCTTCCAATCCAACCACAAACAA
161 +---------+---------+---------+---------+---------+---------+---------+---------
TAGTCTGTTGCAGTCCACCCTCCTGAACCTGCCTTTTCATCTTCCAGTTCTGGTTGGAGAAGGTTAGGTTGGTGTTTGTT

AAAATCAGCCAATATGTCCGACTTCGAGAACAAGAACCCCAACAACGTCCTTGGCGGACACAAGGCCACCCTTCACAACC
241 +---------+---------+---------+---------+---------+---------+---------+---------
TTTTAGTCGGTTATACAGGCTGAAGCTCTTGTTCTTGGGGTTGTTGCAGGAACCGCCTGTGTTCCGGTGGGAAGTGTTGG

CTAGTATGTATCCTCCTCAGAGCCTCCAGCTTCCGTCCCTCGTCGACATTTCCTTTTTTTTCATATTACATCCATCCAAG
321 +---------+---------+---------+---------+---------+---------+---------+---------
GATCATACATAGGAGGAGTCTCGGAGGTCGAAGGCAGGGAGCAGCTGTAAAGGAAAAAAAAGTATAATGTAGGTAGGTTC

Primer 1: 5 G G A C C G C G G G G C A G G A 3

Primer 2: 5 C T T G G A T G G A T G T A A T 3

b) When using the Dideoxy Chain Termination method for sequencing DNA you include only a small
amount of ddATP, ddCTP, ddGTP, and ddTTP.

Give two differences between the ddATP used in the sequencing reaction and the regular dATP.
The ddATP would be missing the 3 OH that is found on dATP and it would have some marker that
will allow visualization.

Explain why your the Dideoxy Chain Termination method for sequencing DNA would not work if
you included too much ddATP, ddCTP, ddGTP, or ddTTP.
If you included too much ddATP, ddCTP, ddGTP, or ddTTP, then each elongation reaction would
incorporate a ddNTP very early in the reaction. This would cause all of the products to terminate
near their 5 end and you would never generate products that would allow you to determine the
sequence of the end of the template.
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7.01SC Fundamentals of Biology

Fall 2011

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